| Objective:The objective of this study is to explore the mechanism of Tongxie Anchang Decoction in treating irritable bowel syndrome with diarrhea(IBS-D)through clinical and basic studies,and to provide scientific basis for its further treatment of IBS-D.Methods:(1)Clinical research:Chapter 1:Collect blood samples from healthy people and IBS-D patients,prepare peripheral blood mononuclear cells(PBMCs),and conduct Single-cell RNA sequencing(scRNA-seq)to draw immune cell picture of IBS-D patients,find key immune cell subsets,and explain the pathogenesis of IBS-D based on changes in immune spectrum.Chapter 2:After 4 weeks of treatment with Tongxie Anchang Decoction for IBS-D patients,the venous blood of patients was drawn to prepare PBMC.The characteristics of immune spectrum changes of IBS-D patients before and after treatment with Tongxie Anchang Decoction were detected based on scRNA-seq,in order to initially reveal the immune regulatory effect of Tongxie Anchang Decoction on IBS-D patients.(2)Basic research:Experiment 1:The model of IBS-D mice with liver depression and spleen deficiency syndrome was reproduced by restraint stress and gavage of senna leaves.The mice were gavaged with low,medium and high doses of Tongxie Anchang Decoction and Pinaverium Bromide Tablets respectively for 2 weeks.The body weight,stool properties,stool water content and colon tissue of the mice were observed for pathological morphology to evaluate the drug efficacy of Tongxie Anchang Decoction in treating IBS-D.Experiment 2:The IBS-D mice with liver depression and spleen deficiency syndrome were treated with different doses of Tongxie Anchang Decoction by gavage.The changes of immune cells in the mesenteric lymph nodes of the mice were detected by flow cytometry,and the levels of TNF-α,IL-6,IL-17 and IL-10 in the colon of mice were detected by ELISA to reveal the immunomodulatory effect of Tongxie Anchang Decoction on IBS-D mice.Experiment 3:Collect the fresh feces of mice in each group after the treatment,detect the changes of intestinal flora types and abundance of mice in different groups by 16S rRNA sequencing,and evaluate the effect of Tongxie Anchang Decoction on intestinal microecology of IBS-D mice with liver depression and spleen deficiency syndrome.Experiment 4:After replicating the IBS-D mouse model,the mice were treated with drugs for 4 weeks.The abdominal withdrawal reflex(AWR)score of the mice was recorded by balloon dilatation stimulation.The expression levels of 5-HT,SP,CGRP,VIP and in the serum of the mice were detected by ELISA,To evaluate the regulatory effect of Tongxie Anchang Decoction on visceral hypersensitivity in IBS-D mice.Results:(1)Clinical research:Chapter 1:After cell cluster analysis of all effective 55839 cells,a total of 12 cell clusters were determined.After the cell clusters were annotated according to cell specific marker genes,eight types of cells were obtained,including CD4+initial T cells,central memory CD4+T cells,CD8+NKT cells,natural killer cells,CD4+responsive T cells,immune B cells,red blood cell like and red blood cell precursor cells,and CD56hiCD62L+NK cells.The difference analysis between groups found that compared with healthy subjects,five types of immune cells in the peripheral blood of IBS-D patients,including CD8+NKT,NK,CD4+CMT,immune B and CD56hiCD62L+NK cells,changed.The function enrichment analysis of differential genes of different cell groups showed that the mechanism might be related to intestinal bacterial infection,differentiation and activation of T helper cells,inflammatory reaction,etc.,indicating that there was significant variation in the immune spectrum of IBS-D patients.Chapter 2:After cluster analysis of all effective 55871 cells,a total of 12 cell groups were determined,which were finally annotated as 10 types of cells:CD4+initial T cells,CD4+ effector T cells,central memory CD4+T cells,CD8+NKT cells,red blood cell like and red blood cell precursors,natural killer cells,CD8+ initial T cells,regulatory T cells,CD56hiCD62L+ NK cells,and immune B cells.The differential analysis found that the therapeutic effect of Tongxie Anchang Decoction may be related to five types of cells,including CD4+CMT cells,Treg cells,NK cells,CD56hiCD62L+NK cells and immune B cells.The main functions of differential genes of differential cell populations are concentrated in the activation and differentiation of immune cells,intestinal infection,inflammation and other related pathways.(1)Basic research:Experiment 1:The IBS-D mice in the model group showed different degrees of depression,increased resistance during gastric perfusion,dry and disordered hair,less movement and aggregation,loose stool texture or other stool characteristics changes,and the mice gained weight,Bristol fecal character score and fecal water content increased.After 2 weeks of drug treatment,the general condition of mice in each dosage group of Tongxie Anchang Decoction and Pinaverium Bromide Tablets group was significantly improved compared with the model group,and the weight of mice increased to varying degrees.Bristol stool character score and stool water content were improved compared with the model group,among which the effect was more significant in the middle dosage group.The HE staining of colon tissue in mice in each group showed no significant difference in colon histomorphology.Experiment 2:In the model group,the proportion of Th1 cells and Th17 cells in the mesenteric lymph nodes of mice increased significantly,while the proportion of CD3+CD4+T cells,Th2 cells and Treg cells decreased significantly,the levels of TNF,IL-6 and IL-17 in colon tissue increased,and the level of IL-10 decreased.After 2 weeks of drug treatment,The percentage of CD3+CD4+T cells in each dosage group of Tongxie Anchang Decoction increased in varying degrees,and the high dosage group significantly increased compared with the model group;Th1 and Th17 cells of mice in each dosage group of Tongxie Anchang Decoction decreased significantly,while Th2 and Treg cells increased significantly;The pro-inflammatory factor of mice in each dose of Tongxie Anchang Decoction group decreased,while the anti-inflammatory factor IL-10 increased.The results showed that Tongxie Anchang Decoction could regulate the immune imbalance of IBS-D mice.Experiment 3:The gut microbiome of IBS-D mice in the model group were disordered.Tongxie Anchang Decoction treatment increased the α diversity and β diversity of gut microbiome in mice.At the portal level,drug treatment increased the abundance of Verrucomicrobiota.At the generic level,after 2 weeks of treatment with Tongxie Anchang Decoction,the abundance of Akkermansia was significantly increased.The results showed that Tongxie Anchang Decoction could alleviate the imbalance of gut microbiome in IBS-D mice.Experiment 4:The AWR scores of the model group mice were significantly increased at 0.25,0.35,0.5,0.65 ml gas pressure levels,the contents of serum 5-HT,SP,CGRP,VIP were increased.After 2 weeks of drug treatment,compared with the model group,the AWR scores of mice in the Pinaverium Bromide Tablets at 0.25,0.35,0.5,0.65 ml gas pressure levels were significantly reduced,the AWR scores of mice in the middle dose and high dose groups at 0.25,0.35,0.5,0.65 ml gas pressure levels were also significantly reduced,and the AWR scores of mice in the low dose group at 0.25,0.35,0.5 ml gas pressure levels were significantly reduced.Serum 5-HT,CGRP,VIP and SP of mice were decreased to varying degrees compared with the model group.The results showed that Tongxie Anchang Decoction could improve visceral hypersensitivity of IBS-D mice.Conclusions:Tongxie Anchang Decoction has significant clinical effect on IBS-D,whose mechanism is related to regulating intestinal immune function,regulating the homeostasis of beneficial gut microbiome such as intestinal Ackermann,and improving visceral hypersensitivity. |
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